TECHNOLOGY CORE 2 (TC2) - CRYO-ELECTRON MICROSCOPY (CHENG. CORE LEADER) A. SPECIFIC AIMS For large complexes, it is essential to bridge the size scales from the atomic-resolution structures of individual subunits to the organization of supramolecular assemblies. EM is ideally suited for this purpose, and every HARC Center project has a single particle cryo-EM component to examine the structures of several HIV/host protein complexes. Consequently, TC2 is designed to enable a broad attack on HIV regulatory complexes by EM. The success of this approach depends on the capacity to align and average a large number of images from molecules with identical structures. We will deal with two key technical issues: that the size of the complexes must be large enough to computationally align images taken from samples embedded in vitreous ice; and that the structural.heterogeneity inherent to many macromolecular assemblies must be adequately dealt with. We will advance the current limitations of cryo- EM to enable studies of small HIV/host complexes with expected structural heterogeneities. We will combine approaches based on cryo-electron tomography (cryo-ET) with those developed for conventional single particle analysis. In Aim 1, we will establish key experimental methods by studying the trimeric HIV envelope glycoprotein gp120/gp41 and its complex with the host receptor CD4 by conventional single particle methods. In Aim 2, we will use cryo-ET to study the trimeric gp120/gp41/CD4 complex. In Aim 3, we will apply the methods developed to other HIV/host protein-nucleic acid complexes, including IN/DNA, Tat-TAR-P-TEFb/RNAP II/DNA, and Rev/RRE/Crm1 complexes. An overall aim of TC2 is to develop new methods that will bridge the gap between macromolecular assemblies that can be determined by cryo-ET and cryo-EM.